Human cells possess a strand-specific mismatch repair system that is a homolog of the bacterial and yeast pathways as judged by similarities in specificity, mechanism, and involvement of MutS and MutL-like activities. As in microbial systems, human mismatch repair mutations confer hypermutability. Genetic defects in the human repair system are the cause of hereditary nonpolyposis colon cancer, have been implicated in a subset of sporadic tumors that arise in a variety of tissues, and also confer resistance to certain chemotherapeutic agents, including DNA methylators and cisplatin. Clarification of the molecular nature of the human mismatch repair reaction is therefore relevant to both the origin and treatment of human malignancies. We and others have identified six activities that are involved in the human repair reaction: the mismatch recognition activities MutS-alpha (MSH2.MSH6 heterodimer) and MutS-beta (MSH2.MSH3 heterodimer), MutL-alpha (MLH1.PMS2 heterodimer), PCNA, RPA, and DNA polymerase delta. One of our major goals for the requested extension of this work will be the identification and isolation of other components of the repair system, with emphasis on helicase and/or exonuclease activities involved in the excision step of the reaction. This fractionation work will also include the isolation and characterization of several novel MutL-like activities that have been identified in extracts derived from HeLa cells and certain MLH1- deficient tumor and drug-resistant cell lines. The second aim of the work will be to further clarify the molecular roles of the required activities in the mismatch repair reaction. Initial studies will emphasize the assembly of the initiation complex, including protein- protein interactions, and the role of ATP in initiation complex formation and evolution. Should excision activities become available during the course of the project, the molecular studies will be extended to include these activities.